Cellular injury resulting from freezing and thawing has been studied for over sixty years. Under slow cooling conditions ice will form outside of the cell, concentrating solutes and creating an osmotic flux. Cells with less permeable membranes will incur increasing osmotic pressure resulting in intracellular ice formation and cell rupture if dehydration of the cell cannot occur. However, rapid dehydration may also be lethal to the cell and cryoprotectants are employed to mitigate cellular damage. Cryoprotectants are cytotoxic, and the mechanism of intracellular ice formation and subsequent cell death are not well understood. While the formation of intracellular ice correlates with cell death, it does not directly kill cells. Rather, the process of ice recrystallization (a form of ice crystal re-modeling that occurs during warming), is believed to be a significant factor contributing to cell death. The importance of ice recrystallization as a mechanism of cellular damage is supported by the fact that (1) freeze-tolerant organisms inhabiting sub-zero environments produce large quantities of recrystallization inhibitors in vivo to ensure survival and (2) ice recrystallization damages cell membranes during cryopreservation.
Stem cell and regenerative therapy using cryopreserved umbilical cord blood is hampered by decreased cell function and viability after thawing. Consequently, improved cryopreservation protocols that increase the yield of viable and functional cells are urgently required. Dimethyl sulfoxide (DMSO) is currently regarded as the “gold standard” for cryopreservation of stem cells and umbilical cord blood. While all cryoprotectants are potentially cytotoxic in vitro, DMSO has exhibited significant cytotoxic effects in the clinical setting. Various biopolymers have been explored as an alternative to DMSO, but fail to provide the high cell viabilities observed with DMSO or glycerol. Similarly, various sugars (mono- and disaccharides) have also been investigated as cryoprotectants. However, the structure of the carbohydrate, the freezing protocol, cell type and reported cell viabilities vary dramatically between studies making it difficult to ascertain the true ability of these compounds to protect cells against cryo-injury. To date, a viable alternative to DMSO, has not yet been identified.
Improved cryopreservation compositions and methods have the potential to revolutionize solid organ transplantation by allowing organs such as livers and kidneys to be successfully preserved and transported more widely.
The mechanism of recrystallization (both with inorganic materials and ice) has been studied and several models have emerged.
Biological antifreezes (BAs) are a very interesting class of molecules comprised of antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) which protect organisms inhabiting sub-zero environments from cryo-injury and death. However, attempts to utilize BAs as cryoprotectants have been met with limited success. This is largely because BAs exhibit thermal hysteresis (TH) activity, meaning they have the ability to selectively depress the freezing point of a solution below that of the melting point. The TH activity associated with these compounds is a result of irreversible binding of BAs to the surface of ice, exacerbating cellular damage at cryopreservation temperatures. This is unfortunate as BAs are also potent inhibitors of ice recrystallization.
During the last decade, the rational design of novel compounds based on BAs has featured prominently in the literature. However, only a few of these molecules have the ability to inhibit ice recrystallization, limiting compounds with potent ice recrystallization inhibition activity to BAs (native AFPs and AFGPs), synthetic analogues of AFGPs and polyvinyl alcohol (PVA). It has been demonstrated that C-linked AFGP analogues 1 and 2, shown below
possess “custom-tailored” antifreeze activity. They are potent inhibitors of ice recrystallization but do not exhibit TH activity. The cryoprotective ability of C-linked AFGP analogues 1 and 2 has also been assessed, and analog 1 was found to be as effective as a 2.5% solution of DMSO for the cryopreservation of human embryonic liver cells. It has also been demonstrated that simple mono- and disaccharides are moderate inhibitors of ice recrystallization and that inhibition of ice recrystallization during cryopreservation with human embryonic liver cell lines and human umbilical cord blood leads to increased cell viability after thawing. While these simple carbohydrates classify as small molecules, they are not potent inhibitors of ice recrystallization. Consequently, small molecules exhibiting potent IRI activity are very attractive, but efforts to design such molecules have been impeded because the structural attributes necessary to inhibit ice recrystallization remain unknown.